Polyacrylamide gel electrophoresis (PAGE) is protein analysis technique useful for separation, identification, and purity and size determination of proteins. Particularly, in sodium dodecyl sulfate-PAGE (SDS-PAGE), proteins are strongly bound to SDS, an anionic surfactant, in a ratio of 1.4:1, so that the protein molecules have negatively charged surfaces and are separated only based on their sizes. The SDS-PAGE is simple and has a high resolution, and thus is widely used as a method for protein analysis (see, A. T. Andrews, Electrophoresis, pp. 1-58).
Most proteins to be analyzed are colorless, and thus require an adequate means for detection. Detection methods that have been reported to date include organic dye staining, silver staining, fluorescence staining and background staining.
Organic dye staining is a detection method of proteins, which includes staining protein bands with an organic dye such as Amido Black 10B, Ponceaus S, Fast Green FCF, or Coomassie Brilliant Blue R (CBBR) [see, J. of Chromatography A, 698, 123-143 (1995)]. Particularly, staining with CBBR is relatively simple and cost effective, and thus is commonly used. However, CBBR staining requires a long time (8-12 hours) to perform staining and destaining, and provides a relatively low sensitivity [sensitivity of 50 ng for bovine serum albumin (BSA)]. Silver staining provides the highest sensitivity among non-radiation detection methods, and relies on deposition and reduction of silver [see, J. of Chromatography A, 698, 123-143 (1995)]. Although the silver staining method provides such a high sensitivity that the method allows detection of up to 0.1 ng, i.e., femtogram scales, the method requires complicated and multi-step processes. Fluorescence staining is a detection method including labeling proteins to be analyzed with a fluorescence-sensitive dye. Although the fluorescence staining method provides a high sensitivity, the method requires complicated process, ultraviolet (UV) irradiation, and precise instruments for quantification, and thus is not cost effective.
Background staining is limited onto SDS-containing gels, and includes forming precipitates on the surface of gels except protein bands with a metallic salt. Although the background staining method allows detection in a short time and has improved applicability, the method shows a short retention time of staining, and has difficulty in storage [see, Anal. Biochem. 174, 157-167 (1988)]. Therefore, there has been a continuous demand for a rapid and simple detection method of proteins on polyacrylamide gels, which provides a higher sensitivity as compared to the prior detection methods.
Eosin Y (Acid Red 87, C. I. 45380) is a water-soluble yellowish red dye emitting green fluorescence (λmax=514 nm) and is represented by the following Formula 1:

Phloxine B (Acid Red 92, C. I. 45410) is a water-soluble orange dye represented by the following Formula 2:

Both eosin Y and phloxine B contain a carboxyl group, and thus are classified as acidic dyes like CBBR. In histochemical staining, eosin Y is the most commonly used counter-dye against alum hematoxylin for H&E (heamtoxylin & eosin Y) staining method. It is also used as a counter-dye in Gram-Weigert method for Gram-positive bacteria, and it constitutes Papanicolaou's EA solution for staining exfoliated cytologic specimens. Further, it is used in Romanowsky staining method (see, Lillie, R. D. 1977, H. J. Conn's Biological Stains, 9th edition, p. 335-344, Williams Wilkins, Baltimore, Md.). Eosin Y was used in staining proteins on polyacrylamide gels for the first time (see, Lin, F., W. Fan and G. E. Wise, 1991, Eosin Y staining of proteins in polyacrylamide gels, Anal. Biochem. 196: 279-283). Phloxine B is a non-toxic dye used as an additive for foods, pharmaceuticals and cosmetics, and is used as a substitute for Gram staining (see, Rasooly R. 2007, Phloxine B, a versatile bacterial stain. FEMS Immunol. Med. Microbiol. 49: 261-265).
The present inventors developed a method for detecting proteins on a polyacrylamide gel, which includes staining the polyacrylamide gel with a composite dye composition containing an acidic organic dye and a basic organic dye, as well as a composite dye composition for use therein, and obtained Korean Patent No. 298,471 and U.S. Pat. No. 6,277,643 therefor.
Further, they developed a method for quantifying a protein, which includes reacting 0.001-0.006% (w/v) of eosin Y with the protein in an acidic solution with a pH of 2.2-3.8 to form a protein-eosin Y complex, and measuring the absorbance of the protein-eosin Y complex at a wavelength of 530-555 nm, and obtained Korean Patent No. 303,102 therefor. However, the aforementioned method employs a composite dye composition containing two or more organic dyes, or relates to the quantification of proteins with eosin Y. Thus, those methods are irrelevant to the detection of a protein disclosed herein, which includes detecting the protein on a polyacrylamide gel by background staining with an organic dye composition containing eosin Y or phloxine B.